The long-term objective is to characterize structure-function relationships of the two simian virus 40 (SV40)-specific membrane antigens defined as surface (S) and perinuclear (U) antigen. In these studies, SV40-transformed lymphocytes (GD248) will be used initially; later we will also employ a human SV40-transformed lymphoid cell line. The projects planned can be subdivided into three major categories: 1. Quantitative distribution of the two antigens in different subcellular membranes. 2. Spatial distribution and neighborhood relationships of these antigens within the membrane. 3. Their chemical characterization in different membranes to assess the mechanisms of host cell modification of one original SV40 gene product. Working with lymphoid cells neoplastically transformed with SV40 we plan: (a) To establish a human lymphoid cell line derived from T- or B-cells transformed in vitro with SV40; (b) to prove whether both S- and U-antigens are expressed on the cell surface; (c) to define their quantitative distribution in the outer envelope of the nuclear membrane (domain of U-antigen), in membranes of the endoplasmic reticulum and in the plasma membrane (domain of S-antigen); (d) to analyze the spatial distribution of S- and U-antigens across the membrane using controlled proteolysis and labeling techniques from both sides of the membrane; (e) to specifically investigate the neighborhood relationships between SV40 and HL-A antigens in plasma membranes of human lymphoid cells using (1) hydrophilic and hydrophobic photoactivated reversible cross-linking agents followed by (2) immunochemical analyses of cross-linked membrane proteins/glycoproteins with sera specific for HL-A, S- and U-antigens; (f) to isolate SV40-induced S- and U-antigens by preparative isoelectric focusing on the basis of their extreme isoelectric points, pI 4.5 and pI 4.7 respectively; (g) to chemically characterize the protein and carbohydrate moieties of the two groups of antigens using (1) N-terminal amino acid analyses, (2) peptide/glycopeptide mapping, (3) amino acid analyses of peptides (glycopeptides), and (4) bulk and sequential analyses of the oligosaccharide residues.